Therapeutic methods which utilize oligonucleotides as active agents are based on a number of end strategies. The earliest concept in this group of strategies appears to be the "antisense" approach wherein the oligonucleotide is designed to be the antisense counterpart of an mRNA transcript and is thus expected to interrupt translation of a gene which has an undesired effect in the cell. More recently, it has been found that oligonucleotides complex with duplex DNA to form triplexes in a sequence-specific manner according to what have been designated as "GT" and "CT" interaction modes. Thus, not only could mRNA transcripts serve as targets, transcription could be interrupted by targeting the duplex DNA. More recently still, it has been found possible to utilize the polymerase chain reaction (PCR) to amplify selectively oligonucleotides that empirically preferentially bind to targets of diverse molecular structure, including proteins and lipids. While the rules for sequence specificity for this type of targeting have not been elucidated (and perhaps there are none), this approach, at least in theory, permits targeting of any desired substance by the properly selected oligonucleotide. The ability to obtain specifically binding oligonucleotides in this way has expanded the possibilities for oligonucleotide therapy in that it may be possible to design oligonucleotides to target substances that reside at the cellular surface.
Nevertheless, a large number of desired targets, including all of the mRNA and double-stranded DNA targets are intracellular. A major barrier to the application of oligonucleotide therapy techniques to living systems has been the inability of oligonucleotides to cross cellular membranes. Native oligonucleotides are highly ionic, indeed negatively charged, high molecular weight materials. Such materials do not readily transit the lipophilic cell membrane.
Numerous publications have appeared that describe inhibition of gene expression by exogenously added oligomers in various cell types (Agrawal, S., et al. Proc Natl Sci (1988) 85: 7079-7083; Uhlmann, E., et al., Chem Revs (1990) 90: 583-584). However, oligomers added directly to cells enter the cellular cytoplasm at a low efficiency, at best, as described below. Many of the apparent sequence-specific effects that have been described are likely to be due to effects on cellular activity that do not arise from binding of the oligomer to target nucleic acid sequences in cytoplasm or nucleoplasm. In the case of RNA antisense sequences generated in situ that are complementary to a target sequence or in cell-free in vitro systems with exogenously added oligomers, gene specific effects do appear to occur by binding of the oligomer to the target sequence (Oeller, P. W., et al., Science (1991) 254: 437-439; Joshi, S., et al., J Virol (1991) 65: 5524-5530; Haeuptle, M-T., et al., Nucl Acids Res (1986) 14: 1427-1448) .
It has been generally assumed that oligomers containing the native phosphodiester linkages enter cells by receptor-mediated endocytosis (Loke, S. L., et al., Proc Natl Acad Sci USA (1989) 86: 3474-3478; Yakubov, L. A., et al., (ibid.) 6454-6458). Subsequent studies appear to show that oligomers with modified internucleotide linkages that may mitigate the presence of negative charges also enter the cells through specific receptors, rather than by passive diffusion (Akhtar, S., et al., Nucleic Acids Res (1991) 19: 5551-5559; Shoji, Y., et al., (ibid.) 5543-5550). Entry of oligomers into cells by either receptor mediated endocytosis or by other mechanisms results in their localization into intracellular endosomes or vesicles. Thus, entry of oligomers into cellular cytoplasm or nucleoplasm is prevented by the membrane barrier surrounding these subcellular organelles (Shoji, Y. et al., (ibid) 5543-5550). Because of the low rate of such endocytosis, it has been necessary to attempt to protect the oligonucleotides from degradation in the bloodstream either by inclusion of these materials in protective transport complexes, for example with LDL or HDL (deSmidt, P., et al., Nucleic Acids Res (1991) 19: 4695-4700) or by capping them with nuclease-resistant internucleotide linkages (Hoke, G. D., et al., (ibid.) 5743-5748).
No progress has been reported in designing oligonucleotides which are capable of passive cell membrane diffusion, so as to be able to enter cells rapidly across cellular membranes to interact with intracellular targets. Those factors related to molecular characteristics which determine the diffusion coefficients of molecules in general have, however, been extensively studied. See, for example, Stein, W. D., in "New Comprehensive Biochemistry", Vol. 2 (Membrane Transport), Elsevier/North Holland Biomedical Press (1981), pp. 1-28; Lieb, W. R., et al., Nature (1969) 224: 240-243. It has been concluded that the distribution constant for a particular substance between the lipophilic membrane and an external aqueous phase is a direct function of the partition coefficient of the material between octanol and water times the molecular weight of the material of interest raised to an appropriate negative power characteristic of the membrane. As the appropriate negative power for, for example, red blood cells is about -4, it appears that high molecular weight substances must have hopelessly low distribution coefficients between cellular membrane and the external environment, even if their partition coefficients for octanol:water are quite high. The validity of this relationship for various small molecules, however, appears to be substantiated by experiment (Hansch, C., et al., J Pharm Sci (1972) 61: 1-19; Walter, A., et al., J Membrane Biol (1986) 90: 207-217).
The partition coefficient for native DNA or RNA is relatively low; less than 0.0. DNA modified by synthesis of 2-methoxyethylphosphoramidite internucleoside linkages in place of the phosphodiester linkage eliminates the negative charge associated with the internucleotide linkage, which increases the hydrophobicity of DNA. However, the octanol-water partition coefficient (Log P.sub.oct) remains less than 0.0 (Dagle, J. M., et al., Nucl Acids Res (1991) 19.: 1805-1810). Increased Log P.sub.oct values for 2-methoxyethylphosphoramidite-modified DNA were assayed by measuring the partitioning of radiolabeled DNA in an octanol-aqueous buffer system. Increased Log P.sub.oct was correlated with increased retention time on reversed-phase HPLC columns (Dagle, J. M., et al., Nucl Acids Res (1991) 19: 1805-1810). Other DNA analogs, such as methylphosphonates or thioates, or DNA with lipophilic adducts (Severin, E. S., et al., Adv Enzyme Regulation (1991) 31: 417-430) that are described in the literature are similarly expected to have Log P.sub.oct values less than 0.0. Oligomers containing high levels of both modified bases and internucleotide linkages have not been described.
It has now been found that by appropriate design of their molecular features, oligonucleotides can be modified from their native forms so as to permit their passive diffusion across cellular membranes, despite the high molecular weights inherent in these molecules. A standard oligonucleotide dimer with two linkage groups has a molecular weight of about 650 Daltons. The relevant factor this generates in determining distribution between membrane and aqueous medium is thus very small, which indicates that such a molecule is essentially impermeable to cell membranes. The dimers and higher molecular weight oligonucleotides of this invention are, however, capable of passive diffusion into cells. Oligonucleotide dimers, as used herein, are generally comprised of two bases and either one or two phosphodiester internucleotide linkage groups, with one linkage found between the nucleosides and a second linkage which is usually attached to the 5'-terminal hydroxyl group. Such dimers can have a third linkage attached to the 3' hydroxyl group.
The oligonucleotides of the invention, when fluorescently labeled and utilized as agents for visualizing cells or subcellular structures, are characterized by a log value of the distribution coefficient between octanol and water of about 0.0-2.5. Such oligonucleotides are capable of efficiently traversing cell membranes and have a minimum solubility in water or aqueous media of at least 10 nM, preferably 50 nM. The minimum solubility requirement is based on the minimum concentration of fluor required by current fluorescent microscopes for visualizing the label. The oligonucleotides of the invention, when utilized as (i) agents that bind to intracellular or extracellular structures such as proteins or nucleic acids, or (ii) labeled compounds to detect or visualize cells, cell membranes or subcellular components in tissue samples, intact cells or in cell lysates, are characterized by a log value of the distribution coefficient between octanol and water of about 0.0-2.5. Such oligonucleotides also have a minimum solubility in water or aqueous media of at least about 0.001 .mu.g/mL.
Some of the oligonucleotides of the invention were found to bind to specific subcellular components such as endoplasmic reticulum or mitochondria. Because of this, permeation-competent oligonucleotides that are fluorescently labeled can be used to directly visualize live cells or cell components in cell lysates. The aspects of the compounds that confer subcellular component-specific binding on the oligonucleotides of the invention are believed not to reside in the fluorescent moiety that is attached to the compound. However, the same oligonucleotides, either containing the fluorescent label or without the label can be synthesized utilizing, say, .sup.32 P instead of the normal nonradioactive phosphorus isotope. Any other appropriate radiolabel can also be utilized according to conventional methods. Such radiolabeled oligonucleotides would retain their cell component-specific binding properties, but need not be directly visualized. In this case, cells or cell lysates can be specifically bound by the oligonucleotide followed by detection of bound oligonucleotide. Radiolabeled oligonucleotides used in this manner would have a minimum solubility requirement in water or aqueous media of about 0.001 .mu.g/mL in order to be conveniently detected or quantitated by conventional methods such as scintillation counting.